During HIV-1 infection, it is likely that at least four subpopulations of Gag coexist;these subpopulations include Gag polypeptides involved in degradation, assembly, budding, or endocytosis. The coexistence of these populations is problematic to the study of Gag, as characteristics of any one subpopulation cannot be adequately described by examination of total intracellular Gag. I hypothesize that subpopulations of Gag can be distinguished and tracked based on associated host cell factors. Further, I hypothesize that at least two of these subpopulations, assembling and, budding Gag, are temporally linked by a Gag-containing complex that can be isolated. The specific aims of this proposal are to 1) isolate and track the subpopulation of degrading Gag and 2) identify the link between assembly and budding. In Aim 1, a combination of biochemical and pulse-label approaches will be used to determine whether assembly of Gag protects these polypeptides from degradation. Factors that bind to, and thereby distinguish, the degrading Gag subpopulation will be investigated using proteomic and IP/immunoblot. Being able to distinguish degrading Gag from other Gag subpopulations is essential to understanding the temporal and mechanistic relationships between these pools of polypeptides. To illustrate this point, in Aim 2 I propose to identify the link between HIV-1 assembly and budding through the examination of cellular factors involved in these processes. In this aim, I will use a combination of biochemical and ultrastructural techniques to test the hypothesis that the Gag complex that recruits cellular budding machinery is a Gag assembly intermediate. The results from this study will broaden our understanding of how different Gag subpopulations can be isolated and tracked independently and will use these tools to identify the link between assembly and budding. This project will be undertaken working closely with my mentors, Drs. Jaisri Lingappa-and Julie Overbaugh. Multiple cellular proteins are essential for productive HIV-1 infection. PUBLIC HEALTH RELEVANCE: Understanding how these factors promote the mechanics of HIV-1 assembly and virus budding will potentially lead to the discovery of novel drug targets in HIV-1 infection.